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Mouse Gpr17 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Gpr17 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Gpr17 (NCBI Reference Sequence: NM_001025381 ; Ensembl: ENSMUSG00000052229 ) is located on Mouse 18. 2 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000064016). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Gpr17 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-92D21 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a null allele exhibit early differentiation of oligodendroglial progenitor cells and increased myelination without any behavioral or motor deficits. Homozygotes for a different null allele show increased vascular permeability in IgE-dependent passive cutaneous anaphylaxis.

Exon 2 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 2. The size of effective cKO region: ~1297 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele T gRNA region G 5' A 3'

1 2

Targeting vector T G A

Targeted allele T G A

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Gpr17 cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7017bp) | A(24.58% 1725) | C(28.1% 1972) | T(22.66% 1590) | G(24.65% 1730)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr18 - 31948009 31951008 3000 browser details YourSeq 63 377 502 3000 87.1% chr2 + 155330205 155330333 129 browser details YourSeq 63 393 815 3000 69.0% chr10 + 117912787 117912937 151 browser details YourSeq 57 1 439 3000 76.1% chr10 - 91242309 91242702 394 browser details YourSeq 49 377 438 3000 90.4% chr1 - 133440769 133441136 368 browser details YourSeq 47 377 439 3000 94.5% chr2 - 158455615 158455867 253 browser details YourSeq 47 379 438 3000 89.1% chr14 - 21463067 21463125 59 browser details YourSeq 47 397 465 3000 87.4% chr7 + 61591521 61591591 71 browser details YourSeq 46 388 502 3000 94.3% chr10 + 111115325 111115442 118 browser details YourSeq 45 393 544 3000 96.0% chr13 - 5585779 5585937 159 browser details YourSeq 45 379 540 3000 88.2% chr11 - 95729260 95729423 164 browser details YourSeq 44 377 426 3000 94.0% chr9 + 70393904 70393953 50 browser details YourSeq 44 395 503 3000 90.8% chr1 + 125480306 125480416 111 browser details YourSeq 43 370 497 3000 92.0% chrX - 166488312 166488440 129 browser details YourSeq 43 376 425 3000 93.9% chr6 - 97297412 97297461 50 browser details YourSeq 43 182 427 3000 60.0% chr1 - 34801269 34801318 50 browser details YourSeq 42 393 439 3000 95.8% chr11 - 114805187 114805526 340 browser details YourSeq 42 383 426 3000 97.8% chr7 + 35740404 35740447 44 browser details YourSeq 41 380 501 3000 87.3% chr11 + 112768771 112768890 120 browser details YourSeq 40 377 438 3000 82.2% chr10 + 81196287 81196347 61

Note: The 3000 bp section upstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr18 - 31943992 31946991 3000 browser details YourSeq 54 1408 1471 3000 92.2% chr18 - 31946261 31946324 64 browser details YourSeq 54 668 731 3000 92.2% chr18 - 31945521 31945584 64 browser details YourSeq 25 884 912 3000 93.2% chr1 - 28806918 28806946 29 browser details YourSeq 22 2456 2477 3000 100.0% chr7 + 29491240 29491261 22 browser details YourSeq 22 1441 1462 3000 100.0% chr3 + 135950191 135950212 22

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Gpr17 G protein-coupled receptor 17 [ Mus musculus (house mouse) ] Gene ID: 574402, updated on 8-Oct-2019

Gene summary

Official Symbol Gpr17 provided by MGI Official Full Name G protein-coupled receptor 17 provided by MGI Primary source MGI:MGI:3584514 See related Ensembl:ENSMUSG00000052229 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as AI853548 Expression Broad expression in cortex adult (RPKM 9.4), frontal lobe adult (RPKM 8.2) and 23 other tissues See more Orthologs human all

Genomic context

Location: 18 B1; 18 See Gpr17 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (31942999..31949890, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (32102653..32109290, complement)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 1 transcript

Gene: Gpr17 ENSMUSG00000052229

Description G protein-coupled receptor 17 [Source:MGI Symbol;Acc:MGI:3584514] Location Chromosome 18: 31,942,997-31,949,636 reverse strand. GRCm38:CM001011.2 About this gene This gene has 1 transcript (splice variant), 183 orthologues, 16 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Gpr17-201 ENSMUST00000064016.5 5195 339aa ENSMUSP00000063670.5 Protein coding CCDS29114 Q6NS65 TSL:1 GENCODE basic APPRIS P1

26.64 kb Forward strand 31.935Mb 31.940Mb 31.945Mb 31.950Mb 31.955Mb Lims2-210 >protein coding Lims2-202 >protein coding (Comprehensive set...

Lims2-211 >lncRNA Lims2-203 >protein coding

Lims2-201 >protein coding

Lims2-207 >nonsense mediated decay Lims2-205 >lncRNA

Lims2-208 >lncRNA

Lims2-212 >lncRNA Lims2-209 >lncRNA

Lims2-204 >protein coding

Lims2-206 >retained intron

Contigs < AC131761.4 Genes (Comprehensive set... < Gpr17-201protein coding < Myo7b-201protein coding

Regulatory Build

31.935Mb 31.940Mb 31.945Mb 31.950Mb 31.955Mb Reverse strand 26.64 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000064016

< Gpr17-201protein coding

Reverse strand 6.64 kb

ENSMUSP00000063... Transmembrane heli... Low complexity (Seg) Superfamily SSF81321 Prints G protein-coupled receptor, rhodopsin-like

PR01157 Pfam G protein-coupled receptor, rhodopsin-like PROSITE profiles GPCR, rhodopsin-like, 7TM PROSITE patterns G protein-coupled receptor, rhodopsin-like PANTHER PTHR24232

PTHR24232:SF44 Gene3D 1.20.1070.10 CDD cd15161

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 339

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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